Severing the taproot of either juveniles or adults caused the entire aboveground biomass to wilt in a matter of minutes, and the plants eventually died. Severing one lateral root caused spatially aggregated wilting in nine of the 24 adults but no wilting of rosettes in juveniles (Fig. 2a). Wilted tissue did not subsequently recover, and that part of the plant was dead the following year in all nine cases. The piece-wise regression revealed a significant size threshold in the wilting response of 20 rosettes (t34 = 11.34, P = 0.001) below which no wilting occurred and above which over 40% of the plants showed some wilting. A simple linear regression showed that the initial number of rosettes explained 25% of the variation in the number of wilted rosettes (t34 = 11.42, P = 0.002; R2 = 0.25), indicating more tissue loss in larger plants; adding the diameter of the clipped lateral root as a covariate did not improve the correlation (ANCOVA: F2,33 = 5.57, P = 0.008, adjusted R2 = 0.21).
Figure 2. Differences in individuals of Cryptantha flava as a function of plant size. (a) Aboveground response to the loss of hydraulic conductivity when a single lateral root was clipped per individual (n = 12 juveniles and 24 adults). The dashed line represents piece-wise regression, with a breakpoint for plants of 20 rosettes. (b) Xylem vessel lumen diameter (μm), distance to closest xylem vessel (μm) and decayed area of heartwood (mm2) increase with caudex diameter and development (n = 4 juveniles and 6 adults). Note different logarithm axes for the anatomical measurements.
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One day after the dye solution was applied to a lateral root, the dye appeared in all rosettes of juveniles, but was aggregated in a specific group of rosettes in adults (Fig. 3). Posterior examination showed that all stained rosettes in adults belonged to the same rosette module (i.e. the dyed rosettes were all connected to the same branch of the caudex). The staining of all rosettes in juveniles was not driven by the existence of only one module, because several juveniles had two, and both were equally stained. In two large juveniles and two small adults, out of the total number of 32 plants, we detected movement of dye to another module when we saturated the atmosphere of the originally stained modules.
Figure 3. Diagram representing a juvenile (a) and an adult (b) of Cryptantha flava 24 h after a dye solution (acid fuchsin) was fed to one lateral root with a vial. In the case of the juvenile the dye stained all leaf rosettes, but in the adults the dye was concentrated into a specific module, as indicated by the distinctive leaf and flower red coloration. Dashed arrows indicate continuation of the root.
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Leaf water potentials
Leaf water potentials differed significantly between juveniles and adults and among watering treatments (Table S1), but not before the watering occurred. At 18:00 h, there was no significant effect of treatment (F2,131 = 0.42, P = 0.65), developmental stage (t133 = 1.85, P = 0.18) or their interaction (F3,130 = 0.83, P = 0.48) on leaf water potential. After watering, leaf water potentials were higher – less negative – in the full watering treatment than in the partial watering treatment, which were higher than in controls (F2,532 = 8.68, P < 0.001; ψL Full = −0.78 ± 0.03 MPa; ψL Partial = −0.76 ± 0.02 MPa; ψL Control = −0.59 ± 0.03 MPa; mean ± standard error of leaf water potentials in full, partial or control watering treatments, respectively). For juveniles, partially watered plants had higher water potentials than controls only at 22:00 h, whereas partially watered adults also had significantly greater leaf water potentials at midday (Fig. 4a; Table 1). Leaf water potentials were elevated in the full watering treatment, compared with controls, at 22:00 h (t14 = 22.65, P < 0.001) and predawn (t12 = 7.22, P < 0.02) for juveniles, and at 22:00 h (t27 = 20.90, P < 0.001), predawn (t27 = 8.70, P = 0.005) and midday for adults (t29 = 69.07, P < 0.001). Two separate repeated measures ANOVAs, one for juveniles and one for adults, revealed significant effects of watering treatment and time, as well as their interaction. However, when we considered the variation in leaf water potentials attributable to the modules nested within individuals, the model was statistically significant for adults but not for juveniles.
Table 1. Separate repeated measures ANOVA, for juveniles and adults, for leaf water potentials of modules within individuals exposed to three different watering treatments and followed for four time-points
|df||Mean squares||F-ratio||P-value||df||Mean squares||F-ratio||P-value|
| Treatment||2||0.330||6.074||0.004*||2||0.826||10.046||< 0.001**|
| Treatment (individual)||7||0.352||1.849||0.092||14||1.800||3.122||< 0.001**|
| Treatment (individual (module))||5||0.081||0.597||0.703||23||1.683||1.781||0.018*|
| Time||3||3.166||38.794||< 0.001**||3||7.272||58.984||< 0.001**|
| Time × treatment||6||0.589||3.607||0.004*||6||1.999||8.111||< 0.001**|
| Time × treatment (individual)||21||1.251||2.190||0.008*||42||9.456||5.479||< 0.001**|
| Time × treatment (individual (module))||15||0.620||1.519||0.123||69||5.502||1.941||< 0.001**|
Watering only one sector of the plant increased variation in leaf water potential more among modules of adults than among modules of juveniles. In partially watered juveniles, the variance component attributable to modules (Fig. 4b) was 26% before watering, at 18:00 h, 38% at 22:00 h, and 29% the following midday. In contrast, variation in leaf water potential among modules within partially watered adults increased from 6% before watering and 7% at 22:00 h to 28% the following midday. Interestingly, in both partially watered juveniles and adults, the variance component of leaf water potential attributable to modules was zero before dawn when plants are assumed to be at equilibrium with soil water potential (Sperry et al., 1996).
With subsequent application of dye to the root in the watered sector of partially watered plants with multiple modules, the dye always travelled to the module that had previously experienced the highest leaf water potential at midday ( = 46.79, P < 0.001; Fig. 6 & Fig. S1). After the surrounding atmosphere of the stained rosette modules was saturated, no dye moved to other regions of the plant, with two exceptions: in juvenile 4 (Fig. 5), the dye travelled to the only other module, staining the entire plant; in adult 4, dye travelled to one of three previously unstained modules, which had shown the second highest leaf water potential among all the modules of the plant.
Figure 5. Average leaf water potential of modules (± standard error (SE)) for juveniles and adults. After all the leaf water potentials had been measured, the hydraulic conductivities were tracked using green fast FCF (green) or acid fuchsin (red). The dashed line represents the module that showed the dye after 24 h while solid lines represent modules that did not receive dye. The dotted line represents the module to which the dye transferred when the atmosphere of the previously stained module (dashed line) was saturated. Complete experimental results can be found in Supporting Information Fig. S1.
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Unstained modules of partially watered plants operated at similar leaf water potential as modules of control plants. For juveniles, this was true regardless of the time of measurement (t ≥ 3.65, P ≥ 0.08). In adults, unstained modules of partially watered plants did not differ in water potential from controls (t ≥ 3.47, P ≥ 0.07) except at midday when they had higher values than controls (t53 = 13.33, P < 0.001). There was slightly less concordance between stained modules of partially watered plants and fully watered plants. In juveniles, stained modules had lower water potential than fully watered juveniles at predawn (t12 = 5.33, P = 0.04). In adults, the same was true at both predawn (t46 = 10.52, P = 0.002) and midday (t51 = 7.91, P = 0.007).
Xylem vasculature differed between juveniles and adults at three levels (Fig. 2b). First, distance from a random point to the closest xylem vessel increased as a function of caudex diameter (t198 = 127.59, P < 0.0001, R2 = 0.39; Fig. 2b), being nearly 10-fold greater for adults (144.87 ± 1.74 μm) than for juveniles (15.56 ± 2.12 μm; t119.43 = −5.77, P < 0.0001). Juveniles already exhibited some signs of sectoriality because their xylem vessels are grouped in bundles separated by the dense packs of rays of the radial system (Figs 1, 6b). This packing resulted in large distances among the xylem vasculatures of these bundles near the cambium region (Fig. 6d). Secondly, the diameter of the xylem vessels increased linearly with caudex diameter (t501 = 216.83, P < 0.001, R2 = 0.30), and it was almost twice as large in adults (40.47 ± 0.69 μm) as in juveniles (22.17 ± 0.84 μm; t500.11 = −17.82, P < 0.0001). Within individuals, variation in xylem diameter was very low. Thirdly, the area of decayed heartwood in the central region of the caudex increased linearly with caudex diameter (t8 = 35.00, P = 0.004, R2 = 0.85; Figs 2b, 6a–g). Comparisons across plants of different sizes showed that heartwood decomposition took place from the pith outwards, first following the groups of rays described above (Fig. 6c,d). In very large adults, decayed heartwood extended to the surface and resulted in physical fragmentation of the live vasculature (Fig. 6e–g,i).
Figure 6. Developmental series of cross-sections of the caudex of individuals of Cryptantha flava. (a–g) Plastic sections (5 μm) of juveniles and adults organized by increasing size. Missing heartwood tissue caused by putrefaction was photo-edited to black background. As individuals develop secondary growth, the heartwood decays and eventually discontinuities give rise to physically independent modules. (h, i) Paraffin cross-sections (40 μm) showing stained xylem vessels (arrows) of a small and a large juvenile and a small adult in which dyes (fast green FCF and acid fuchsin, respectively) were tracked from a lateral root. Scale bar, 1 mm.
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In caudex cross-sections from adults used in the dye-tracking experiment, the dye was confined to a specific portion of the xylem, demonstrating sectoriality in the distribution of the dye. Staining occurred only in the newer xylem tissue, suggesting that older xylem is not functional (Fig. 6h,i).